Nkx2.5/Csx Represses Myofibroblast Differentiation

Abstract

Myofibroblasts are known to play key roles in wound healing and fibrosis. They are thought to arise de novo from fibroblasts, and are characterized by induction of alpha-smooth muscle actin (alpha-SMA) expression. The homeobox transcription factor Csx/Nkx2.5 is critical in heart development and cardiogenesis by inducing expression of genes associated with cardiomyocyte differentiation. Here, we report a novel repressor activity of Nkx2.5 on myofibroblast differentiation. Because a key marker of myofibroblast differentiation is expression of the alpha-SMA gene, we first scanned its promoter region for possible cis-acting elements. The results show three potential binding sites (designated Nkx2.5 element [NKE]-1, -2, and -3) containing the Nkx2.5/Csx consensus binding motif (5'-TNNAGTG-3'). To determine their functional importance, site-directed mutagenesis directed at these elements individually and in combination indicated that mutation of NKE1 and -3 significantly enhanced alpha-SMA gene promoter activity, whereas mutation of NKE2 did not have a significant effect. The results of gel shift assays confirmed that Nkx2.5 could bind to both NKE1 and -3, but not to NKE2. Consistent with the mutagenesis studies, ectopically induced expression of Nkx2.5 inhibited alpha-SMA gene expression. Analysis of nkx2.5 gene expression indicates that it was significantly induced by basic fibroblast growth factor treatment of isolated lung fibroblasts in vitro. In vivo, lung Nkx2.5 expression was significantly diminished in bleomycin-induced pulmonary fibrosis. These findings are consistent with a novel function for Nkx2.5 as a repressor of alpha-SMA gene transcription, which may be of homeostatic significance as a means of suppressing myofibroblast differentiation in the absence of tissue injury.